Herpes simplex virus type-2 (HSV-2) infections are lifelong, and a leading cause of genital ulcer disease woridwide. The clinical outcome of genital HSV-2 reactivation varies widely from completely asymptomatic to infrequent and short-lasting recurrences to frequent and severe genital ulcerations: reasons forthis variability are not known. Using biopsies of genital skin and mucosa from human volunteers, we have shown that HSV-2 reactivation results in a long-term persistence of local immune responses. CDS T cells persist at the dermal-epidermal junction (DEJ) contiguous to the sensory nerve endings where virions are released. The unique anatomical distribution suggests that local CDS T cells might play a pivotal role in rapid containment of viral infection in the periphery, thus inflijencing the clinical and virologic course of HSV-2 disease in humans. In the last grant cycle, we developed a cell-type specific laser capture microdissection (LCM) method to isolate individual CDS T cells in situ and measure their activity. By using combined approaches of CDS-specific LCM, whole genome transcriptional profiling and TCR repertoire deep sequencing, we can now elucidate associations between tissue resident memory CDS T cells and genital herpes disease severity in humans. In this project, we will investigate the association between HSV-2 disease severity and the quantity, quality and diversity of tissue resident memory CDS T cells at the site of previous HSV-2 recurrence in humans. Our specific aims are: 1) to define whether the anatomic distribution, density, decay kinetics and the antiviral signature genes of tissue resident memory CDS T cells differ in participants with mild versus severe genital HSV-2 diseases; 2) to define whether the T cell receptor (TCR) repertoire dynamics and antigenic specificity of tissue resident memory CDS T cells are associated with genital herpes disease severity. We will obtain sequential biopsy tissues from patient cohorts with distinct disease outcomes: mild disease, defined as recurrence rate < 2 episodes per year and severe disease as recurrence rate > 6 episodes per year. This project will define the characteristics of a successful peripheral immune response to HSV-2 that can be harnessed as a potential correlate of immunity during vaccine development. RELEVANCE (See instructions): Genital herpes affects 17% of US population; no cure or vaccine is available. We do not know why some people with this infection have severe disease, and others very mild. We have developed new laboratory methods to study immune cells in samples from people with genital herpes that we will apply to understand the difference between people with mild and severe disease in order to develop an effective vaccine.